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European Biopharmaceutical Review

Identifying Impurities

The growth of the industry has been exponential over the past few years. In 2010, the combined revenue from the top 12 biopharmaceutical products in the US alone was over $30 billion, and it is predicted that the market for these products between 2010 and 2014 will increase to over $160 billion (1).

The manufacture of biopharmaceuticals can be complex due to the fact that they are produced within living cells. With this comes the additional challenge of the immunogenic potential and toxicity of any process-related contaminants. The main source of contamination is from the host cell itself. Cells used for the production and development of biopharmaceuticals include bacteria, yeast, plant and eukaryotic cell types. When used as hosts, they are cultured and kept in a continuous cycle of growth and apoptosis. As part of this cycle, they are continually depositing DNA and proteins into the media surrounding them.

Although these contaminants should be removed in a purification step, this process also has the potential to introduce additional contaminants. Guidelines for the analysis of these contaminants are defined in the International Conference on Harmonisation (ICH) Q6B (2).

DNA Detection

Residual host cell DNA and recombinant DNA (r-DNA) that has been transfected into the host cell is perceived as a principal risk associated specifically with continuous cell line derived products. It is not the DNA per se that is the concern, but the actual coding sequence that may have a potentially tumourigenic effect in humans. The regulatory authorities have therefore set maximum levels of residual host cell DNA per dose within biopharmaceuticals. The World Health Organization (WHO) and the European Medicines Agency have set levels of 10ng per dose, and the Food and Drug Administration (FDA) a maximum of 100pg per dose (3,4). There are currently no set guidelines to the length of the DNA fragments, however it is generally accepted that 90 per cent should be less than 500 base pairs in length. DNA is removed from biopharmaceutical products by sequential purification steps and this is a crucial stage in the production of the final product.

Quantitative Methods

The predominant method for the detection of residual DNA is by quantitative polymerase chain reaction (qPCR). This method has a number of advantages; firstly the analysis is quantitative and relatively rapid, taking around two hours; and secondly the method involves the exponential amplification of a short DNA sequence – approximately 100-200bp in length – by a series of polymerisation cycles. The amplification of the DNA sequence is measured in real time at each progressive cycle using a sequence specific fluorescent probe, with the increase in the amplification of the DNA being directly proportional to the intensity of fluorescence. A sample that contains a lower initial DNA concentration will require more polymerase cycles to increase fluorescence above background level (ct) than that of a sample with a higher initial concentration (see Figure 1). From this data, a standard curve can be generated and the concentrations of unknown samples is calculated (see Figure 2). The limit of quantification (LOQ) can be in the region of 30fg, which is far below the level needed to meet the regulatory guidelines.

A second method for the quantification of DNA uses PicoGreen fluorescent dye. This method utilises the increased fluorescent intensity that is observed when PicoGreen binds to double stranded DNA (dsDNA). The intensity of the signal is directly proportional to the concentration of dsDNA within a sample. This can be compared to a standard curve generated from known standards. The method has a LOQ of 20pg/mL and can detect dsDNA fragments as short as 50bp. The main disadvantage of this type of analysis is that it is non-specific, as any contamination of dsDNA from another source would result in false positives.

A final method utilises DNA binding proteins to quantify single stranded DNA (ssDNA), commonly marketed as a DNA threshold assay. The method involves two sequence independent binding proteins; the first is a biotinylated single stranded binding protein; the second an anti-ssDNA antibody conjugated to the enzyme urease. The sample is incubated with both proteins along with streptavidin to form a protein-DNA complex. This is filtered using a biotinylated membrane, capturing and concentrating the DNA complex on the membrane utilising the strong affinity of streptavidin binding to biotin. The detection involves the addition of urea that is hydrolysed by the urease, producing a change in pH that is proportional to the ssDNA concentration. This system has an LOQ of 2pg. However, the method requires the ssDNA fragments to be longer than 500bp, and it is a much longer process than the two previous methods.

Host Cell Protein

One of the main issues with the production and development of a biopharmaceutical is that of immunogenicity. The two most common causes of an immunogenic reaction are the biopharmaceutical itself or process-related contaminants, including host cell proteins (HCPs) and proteins from the purification stage. Many purification steps may be required and these steps themselves have the potential to co-purify and concentrate these contaminants along with the biopharmaceutical.

All biopharmaceuticals must therefore be purified to reduce these contaminants in order to minimise the incidence of immunogenicity against a biopharmaceutical or its trace contaminants (5). Acceptable levels of these contaminants have not been set by the regulatory authorities and are determined on a case-by-case basis. This is due to many variables including dose, dosage frequency and drug delivery. However, it is generally considered that host cell protein levels should be less than 10μg/mL (6). The lack of purification during development has the potential for the product to fail in the early stages of clinical trials.

The most common detection system for host cell protein contamination is enzyme-linked immunosorbent assay (ELISA). There are a number of commercially available ELISA kits on the market supplied by various companies against many cell lines including Chinese hamster ovary cells and E. coli (7). Most of these ELISA kits are ‘sandwich’ based ELISAs involving two antigen specific antibodies – one bound to a microtitre plate to capture the antigen and a secondary antigen-specific antibody conjugated with an enzyme which forms the antigen sandwich. The secondary antibody facilitates a reaction with a substrate colour change. The level to which the substrate develops is directly proportional to the antigen concentration, and the LOQ of these kits may be in the region of 1ng/mL depending on the kit. Commercially available ELISA kits can be used to determine HCP levels within biopharmaceuticals in the early stages of development. However, the regulatory authorities may request that a process-specific ELISA kit is developed for each individual product before it enters Phase 3 studies.

Antibody Production

The process of developing a process-specific HCP assay is long and complex. This must be started early in the development process to ensure a robust assay is in place prior to Phase 3 trials – a procedure that may take in excess of 12 months. The rate limiting step is raising antigen-specific antibodies with high sensitivity and low cross-reactivity. The initial step of the process is the production of antigen (HCPs) from a mock fermentation process. This involves the fermentation of the cell line in the absence of the gene sequence encoding the biopharmaceutical (8). The media is purified using the same method that was used during production of the product. This will contain antigens without the biopharmaceutical product that may be used to generate polyclonal antibodies. As mentioned, there are a number of purification steps within production, each with a different HCP profile. It must be considered from which of the purification steps the polyclonal antibodies are generated for a representative assay for the analysis of the final product. To raise polyclonal antibodies with a broad range of affinity to the HCP, it may be necessary to adopt a number of immunisation schemes. The immune response should be stimulated against strong and major antigens, as well as the weaker less predominant ones.

One method is to mix the antibodies from previous bleeds with the immunogen and re-immunise. This blocks the major antigens, resulting in a stronger immunogenic response to the weaker antigens – for example passive immunisation (9). Alternatively, a combination of antibodies from previous bleeds can be used to eliminate dominant antigens from the immunogen by immunoaffinity chromatography. This enriches the weaker antigens that may be used in the next immunisation schedule – for example, cascade immunisation (10).

The raised polyclonal antibodies are purified by affinity chromatography and quantified by gel electrophoresis and western blot. The aim of the process is to isolate antibodies with the maximum detection of the HCP contaminants (11). Antibody coverage can be analysed using 2D electrophoresis and western blot. The coverage of 80 per cent in the pI range of four to seven and a molecular weight of 10–120kDa is achievable. There are a number of methods for using these antibodies to develop an ELISA including direct, indirect, competition and sandwich, the choice of which will be dependent on the antibodies and antigen. The main considerations are to ensure a sufficient LOQ that is able to satisfy the regulatory authorities and that the final antibody selection has low cross-reactivity. Both the antigen and antibodies need to be stored for use as reference materials for future assays.

Conclusion

The development and production of biopharmaceuticals is a complicated process and it has moved on a long way since the production of the first recombinant human insulin. As technology has developed, biopharmaceuticals have become increasingly complex. Many patents of the blockbuster biopharmaceuticals will expire over the next few years, with a potential influx of biosimilars across the globe. Reliable analytical data concerning the presence and absence of process-related contaminants will be required to satisfy the regulatory authorities to establish biopharmaceutical approval for both biosimilars and originator drugs.

References

1. Shaughnessy AF, Monoclonal antibodies, British Medical Journal, 345: e8346, 2012

2. Specifications: Test procedures and acceptance criteria for biotechnological/biological products Q6B, ICH Harmonised Tripartite Guideline, 1999

3. Technical Report: Requirements for the use of animal cells as in vitro substrates for the production of biologicals, WHO, 1998

4. Vaccines and related biological products, FDA Briefing Document, 2012

5. Wolter J and Richter A, Assays for controlling host cell impurities in biopharmaceuticals, Bioprocess Int 2(2): pp40-66, 2005

6. Eaton LC, Host cell contaminant protein assay development for recombinant biopharmaceuticals, J Chromat A 705(1): pp105-114, 1995

7. Krawitz DC, Forrest W, Moreno T, Kittleson J and Champion KM, Proteomic studies support the use of multiproduct immunoassays to monitor host cell protein impurities, 6(1): pp94-100, 2006

8. Hoffman K, Strategies for host cell protein analysis, Biopharm, 13(6): pp38-45, 2000

9. Thalhammer J and Freund J, Passive immunization: a method of enhancing an immune response against antigen mixtures, 80(1): pp7-13, 1985

10. Anicetti VR, Immunization procedures for E. coli proteins, Appl Biochem Biotechnol, 22: pp151-168, 1989

11. Eaton LC, Host cell protein assay development for recombinant biopharmaceuticals, J Chromatograph A, 705: pp105-114, 1995


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Andrew Kenyon studied for his BSc in Biochemistry at the University of Salford while working at Sigma Aldrich within their good manufacturing practice facility. He gained a PhD in Molecular Immunology at the University of Aberdeen isolating the first human anti-testosterone antibodies for the treatment of prostate cancer. On completion of his PhD, Andrew joined Wyeth Research for five years, designing monoclonal antibodies to a number of challenging targets. This was accomplished by antibody discovery and engineering across a number of species including humans, sheep and sharks. Most recently, Andrew joined RSSL with a remit to assist in the development of a new biomolecular analysis service.
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